MinireviewCurrent issues with acetaminophen hepatotoxicity—A clinically relevant model to test the efficacy of natural products
Introduction
The search for new drugs and novel therapeutic intervention strategies increasingly includes testing plant extracts and other natural products. In addition, more products of traditional Eastern medicine are being evaluated. Independent of whether extracts are considered or if individual ingredients of a mixture are tested, the pharmacological efficacy of these chemicals needs to be investigated. For compounds that are assumed to have hepatoprotective effects, the model of acetaminophen (APAP) overdose in rodents, especially mice, is one of the most popular experimental in vivo systems used today (Campos et al., 1989, Chen et al., 2009, Gao and Zhou, 2005, Hau et al., 2009, Hsu et al., 2008, Küpeli et al., 2006, Wang et al., 2010, Wu et al., 2008, Wu et al., 2010, Yuan et al., 2010). The advantage of this model is that APAP is a dose-dependent hepatotoxicant, the experiments are technically easy to perform and, most importantly, it is a clinically relevant model. However, after more than 35 years of research there is substantial information in the literature on mechanisms of APAP hepatotoxicity (Hinson et al., 2004, Jaeschke et al., 2003, Jaeschke and Bajt, 2006, Jaeschke and Bajt, 2010, Nelson, 1990, Nelson and Bruschi, 2001). Some of these mechanisms are well established and others are more or less controversial; some are correct and some are skewed by experimental conditions making them not clinically applicable. In addition, many aspects of APAP-induced cell death and liver injury are still unknown. Thus, the mechanisms of APAP hepatotoxicity are extremely complex and the interpretation of in vivo data is difficult. Unfortunately, the model is being used as a tool by investigators who are not necessarily experts in APAP toxicity leading to frequent misinterpretation of data (Jaeschke et al., 2010). Therefore, the purpose of this review on APAP hepatotoxicity is to discuss some of the established and controversial mechanisms and the potential pitfalls one should be aware of when using this model.
Section snippets
Models of acetaminophen-induced liver injury
To study mechanisms of APAP toxicity, the mouse in vivo or primary mouse hepatocytes are most frequently used. Various strains of mice (outbred or inbred strains) are susceptible to APAP toxicity but some strain differences exist (Harrill et al., 2009). Mice are fasted overnight to reduce hepatic glutathione levels and are treated i.p. with doses of 200–400 mg/kg APAP dissolved in warm saline. Liver injury develops between 3 and 5 h and peaks at 12 h after APAP administration. Fasting allows lower
Metabolic activation of acetaminophen
One of the earliest works on mechanisms of APAP hepatotoxicity demonstrated that a small fraction of the dose is metabolized by the cytochrome P450 (cyp) system to form a reactive metabolite (Jollow et al., 1973, Mitchell et al., 1973a, Mitchell et al., 1973b). The metabolite was identified as N-acetyl-p-benzoquinone imine (NAPQI) (Dahlin et al., 1984). The primary enzyme involved is Cyp2E1, but others appear to have a role (Thummel et al., 1993, Wolf et al., 2007, Zaher et al., 1998).
Protein adducts of acetaminophen and oxidant stress
The early “protein binding” hypothesis was repeatedly questioned because the overall fraction of the administered dose that ends up covalently bound to cellular proteins is small and certain interventions appear to be able to separate protein binding from cell injury (Jørgensen et al., 1988). As a result, a competing hypothesis was introduced. Wendel and coworkers hypothesized that reactive oxygen generated during the metabolism of APAP can cause lipid peroxidation (LPO), which may be the
Sources of reactive oxygen and reactive nitrogen species
APAP overdose causes an oxidant stress (Jaeschke, 1990) and peroxynitrite formation (Hinson et al., 1998). The increase in tissue glutathione disulfide (GSSG) levels in vivo, as a specific marker for hydrogen peroxide, is caused by a selective accumulation of GSSG in mitochondria (Jaeschke, 1990, Knight et al., 2001). This suggests that enhanced amounts of superoxide are being generated by the electron transport chain and released into the mitochondrial matrix (Fig. 3). This conclusion is
Lipid peroxidation
Due to the involvement of oxidant stress, LPO is a popular hypothesis to explain massive cell death after APAP overdose. Antioxidant function and protection against LPO are probably the most invoked mechanisms of protection by natural products (Campos et al., 1989, Gao and Zhou, 2005, Hsu et al., 2008, Küpeli et al., 2006, Wang et al., 2010, Wu et al., 2008, Wu et al., 2010, Yuan et al., 2010). LPO is a multistep process requiring initiation of a radical chain reaction and propagation through
Necrosis and apoptosis
APAP-induced liver injury is characterized by extensive cell contents release (liver enzymes), cell swelling, nuclear degradation (karyorrhexis and karyolysis) and an inflammatory response (Gujral et al., 2002). These are typical characteristics of oncotic necrosis (Jaeschke and Lemasters, 2003). Thus, it is generally concluded that APAP-induced cell death in vivo (Gujral et al., 2002) and in vitro (Bajt et al., 2004, Kon et al., 2004) is caused by oncotic necrosis (Fig. 4). However, cell death
Innate immune response
The cell contents released after APAP-induced necrotic cell death initiates an inflammatory response with activation of Kupffer cells and recruitment of neutrophils and monocytes into the liver (Laskin, 2009, Laskin and Pilaro, 1986, Lawson et al., 2000) (Fig. 5). It was recently recognized that some of the compounds generally released by dying hepatocytes can stimulate toll-like receptors on macrophages and other non-parenchymal cells and promote cytokine formation initiating an inflammatory
Conclusions
APAP-induced liver injury, especially in mice, is a clinically relevant model that is suitable to test the efficacy of hepatoprotective natural products and other compounds in vivo. Given the extensive knowledge of the mechanisms of APAP-induced liver injury, the model can also be used to investigate mechanisms of therapeutic action. Importantly, though many studies demonstrate an antioxidant effect or protection against LPO with natural products after APAP treatment, this is highly unlikely
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
Research in the authors' laboratory is supported in part by National Institutes of Health Grants R01 DK070195 and R01 AA12916 (to H.J.) and by grants P20 RR016475 and P20 RR021940 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health. C.D. Williams was supported by the “Training Program in Environmental Toxicology” (T32 ES007079-26A2) from the National Institute of Environmental Health Sciences.
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